The regional distribution of acetylcholine (ACh) and cyclic GMP, ACh turnover rates, ACh fractional rate constant, affinity and number of binding sites for (3H) quinuclidinyl benzylate (QNB) were measured in various regions of the rat hippocampus. In coronal section of the hippocampus, both ACh and cyclic GMP content were found to be the highest in sections of the amygdaloid end. Within subsectional areas of coroneal section of the hippocampus (CA-1, CA-3, and dentate gyrus), the cyclic GMP content, the affinity of (3H) QNB specific binding sites were the highest in the CA-3 and dentate gyrus regions, while the density of (3H) QNB specific binding was the highest in the CA-1 area. In contrast, the fractional rate constant for the efflux of deuretiated ACh was higher in the CA 3 area than in the dentate gyrus. This study demonstrates that an enrichment of ACh content, affinity of (3H) QNB binding sites and cyclic GMP content appears to occur in some specific areas of the hippocampus. These findings suggest that the dissection described in the present experiments defines areas with different degrees of cholinergic innervation. Hence such ACh dissection may be useful to study the cholinergic regulation of the cGMP content of some regions of the hippocampus.